Suppression of insulin target gene expression by SecinH3 quantified by real-time PCR

doi:10.1038/nprot.2006.413

Method Article

Suppression of insulin target gene expression by SecinH3 quantified by real-time PCR

https://doi.org/10.1038/nprot.2006.413

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Genetics

Molecular Biology

Biological techniques

ADP ribosylation factors (ARFs)

secinH3

cytohesin

insulin-like growth factor binding protein 1 (IGFBP1)

SecinH3 causes insulin resistance in mouse liver and in cell culture by inhibiting cytohesins that are effectors of the insulin signalling pathway. Stimulation with insulin leads to increased transcription of glycolytic genes, to suppressed transcription of gluconeogenetic genes and IGFBP1 in liver and in HepG2 cells. These insulin effects can be reverted by SecinH3. We quantified gene expression levels by real-time PCR.

10 mg mouse liver (or ca. 10⁶-10⁷ HepG2 cells grown to 75% confluence)

SecinH3 (100 mM in DMSO)

D5 (10 mM in DMSO)

DMSO

insulin (Sigma)

standard cell culture material and media

RNA-extraction kit (Absolutely RNA, Stratagene)

RT-kit (cDNA-Archive kit, Applied Biosystems)

TaqMan probes for assayed genes (Applied Biosystems)

TaqMan MasterMix, 2x (Applied Biosystems)

Standard cell culture equipment

Real-time PCR machine (BioRad)

A: HepG2 cells

ca. 10⁵ HepG2 cells are seeded into wells of a 12-well culture plate and incubated overnight
after washing twice with PBS, the cells are serum starved for 24 hrs
compounds are added in desired concentrations (SecinH3, D5 and Wortmannin or DMSO alone); final concentration of DMSO was 0.2% in our experiment
cells stimulated with 10 nM insulin for 12 hrs
Total RNA extracted with Absolutely RNA kit (Stratagene) according to instructions from manufacturer; elution volume 50 µl

B: Mouse liver

10 mg liver homogenized in lysis buffer and RNA extracted with Absolutely RNA kit (Stratagene) according to instructions of manufacturer; elution volume 50 µl

RT and real-time PCR

RNA concentration determined by photometry
2.5 µg RNA used for reverse transcription with cDNA Archive kit (Applied Biosystems) according to instructions from manufacturer; RT volume: 50 µl
resulting cDNA diluted 1:3 with H₂O
quantitative PCR performed in 10 µl scale with gene-specific TaqMan probes (Applied Biosystems). All expression-levels were normalized to the expression level of β-2-microglobulin.

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Suppression of insulin target gene expression by SecinH3 quantified by real-time PCR

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